Inhibition of FSP1 enhances venetoclax induced cell death in Acute Myeloid Leukemia by the ferroptosis pathway Free

Objective Acute myeloid leukemia (AML) is the most common malignant hematological neoplasm in adults, and the combination of venetoclax, an inhibitor of BCL-2, with hypomethylating agents significantly improves the survival of AML patients. However, resistance to chemotherapeutic agents is a major obstacle to the effective treatment of AML patients. Ferroptosis, a non-apoptotic programmed death mode first proposed in 2012, is expected to be a breakthrough in the treatment of many major diseases, including tumors. The aim of this study was to explore the role and mechanism of ferroptosis in AML venetoclax resistance and to provide a new target for the clinical treatment of refractory relapsed AML patients.

Methods AML cell lines THP-1 and Kasumi-1 were treated with idarubicin (IDA), venetoclax, the ferroptosis inducer RSL3, the inhibitor Ferrostatin-1, and the ferroptosis suppressor protein 1 (FSP1) inhibitor iFSP1. The cell viability was assessed by the CCK-8 assay, and the SynergyFinder was used to assess the drug synergy scores. The expression of ferroptosis-related genes and proteins, such as the iron death markers PTGS2, GPX4, SLC7A11, and FSP1, was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot. Intracellular reactive oxygen species (ROS) levels were determined by flow cytometry staining, and lipid peroxidation was measured using C11-BODIPY 581/591 fluorescent probe. MDA levels were detected using the malondialdehyde (MDA) detection kit. THP-1 venetoclax-resistant (THP-1/Vene) cells and Kasumi-1 venetoclax-resistant (Kasumi-1/Vene) cell lines were constructed to detect changes in the level of ferroptosis occurring in venetoclax-resistant cells after application of RSL3. Synthesize shRNA to stably knock down the expression levels of target genes in cells in vitro. Bone marrow samples from AML patients with venetoclax sensitivity and resistance were collected to observe the expression of ferroptosis related genes in the patients.

Results

• The ferroptosis inducer RSL3 enhances the anti-AML effect of venetoclax: combined treatment with RSL3 (a cystine transporter protein inhibitor that induces ferroptosis) and venetoclax significantly inhibits THP-1 and Kasumi-1 cell viability and promotes venetoclax-induced levels of unstable iron pools, lipid ROS, and apoptosis in AML cells in a synergistic manner. Ferrostatin-1 partially reversed this phenomenon. The expression of ferroptosis-related gene GPX4 decreased and PTGS2 increased in AML cells after treatment with RSL3.

• THP-1/Vene and Kasumi-1/Vene cells were constructed, and the microarray results of AML resistance-related sequencing data were obtained from the NCBI's Gene Expression Omnibus (GSE134589, GSE66525 and GSE52919), which showed significant differences in the expression of the ferroptosis pathway molecules, TLR4, ATG7, FSP1, CYBB, and MYCN, with a high repetition rate. Validation in venetoclax-resistant cell lines and patient specimens showed that FSP1 expression was significantly higher in both resistant cells.

• iFSP1 enhanced the sensitivity of AML-resistant cells to venetoclax: by treating THP-1, Kasumi-1 sensitive and drug-resistant cell lines with iFSP1, a specific inhibitor of FSP1. We observed that iFSP1 significantly inhibited the growth of AML cells in a dose-dependent manner. iFSP1 in combination with venetoclax further reduced the viability of the cells, and enhanced venetoclax sensitivity.

• Inhibition of FSP1 sensitized AML cells to venetoclax induced cell death via the ferroptosis pathway: iFSP1 and venetoclax combined induced increased lipid ROS levels and elevated associated ferroptosis markers in THP-1/Vene and Kasumi-1/Vene cells. shRNA down-regulation of FSP1 expression promoted increased lipid ROS and ferroptosis markers in THP-1 and Kasumi-1 resistant cells. These results suggest that inhibition of FSP1 through activation of the ferroptosis pathway enhances the susceptibility of AML cells to venetoclax.

Conclusion

• Ferroptosis inducers combined with either conventional chemotherapeutic agents or venetoclax inhibit AML cell proliferation and promote ferroptosis occurrence, providing a new therapeutic direction for venetoclax resistance.

• FSP1 inhibition increased the sensitivity of AML cells to venetoclax via the ferroptosis pathway. iFSP1 in combination with venetoclax may provide a favorable strategy for AML treatment.